Methods And Compositions For Regulating Gene Expression

ABSTRACT

In certain embodiments, the disclosure relates to compositions and methods relating to a translation-based gene regulation system that functions in mammalian cells. In certain specific embodiments, the disclosure relates to methods of regulating gene expression via modulating translation termination.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Application 60/727,327, filed Oct. 17, 2005. The entireteachings of the referenced provisional application are expresslyincorporated by reference herein.

FUNDING

This invention was made with government support under Grant Number NIH5PO-HL54785 awarded by National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The ability to exogenously control the expression of genes in mammaliancells has been a powerful tool of biomedical research. In particular,gene regulation technology has played a central role in efforts tounderstand the role of specific gene products in fundamental biologicalprocesses and in both normal development and disease states. It islikely that this form of genetic technology will continue to have impactin a variety of areas of basic research and may enable new therapeuticparadigms, such as the regulated delivery of protein therapeutics. Thetechnology may also have significant future impact upon the safety ofgene therapy strategies.

To date, most of the gene regulation systems commonly utilized are basedon the control of transcription. Despite their considerable utility,these systems possess some significant limitations due to their relianceon chimeric transcriptional transactivators and specialized promoterelements. Such limitations include the requirement for co-introductionof genes encoding the relevant transcriptional transcriptionaltransactivator along with the gene to be regulated, and the inability toprovide for the “on-off” regulation of a gene in the context of its ownendogenous transcriptional control elements.

Thus, there is a need for developing a novel gene regulation system thatdoes not rely on the control of transcription.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for regulatingtranslation of a target protein by suppressing a nonsense mutation whichhas been introduced into a nucleic acid encoding the target protein.

In certain embodiments, the present invention provides a method forinducing expression of a target protein in a host cell. Such methodcomprises contacting the host cell with an effective amount of an agent,wherein the host cell comprises a nucleic acid encoding a targetprotein, wherein the nucleic acid is operably linked to a promoter andhas been modified to contain at least one nonsense mutation downstreamto and in close proximity of the start codon that initiates translationof the target protein, and wherein the agent suppresses the nonsensemutation. A preferred host cell is a mammalian cell (e.g., a humancell). Examples of the agent that suppresses the nonsense mutationinclude, but are not limited to, an aminoglycoside antibiotic or ananalog thereof, such as G418, and an acetylamino benzoic acid compoundor a derivative thereof, such as3-[2-(4-tert-butyl-phenoxyl)-acetylamino]-benzoic acid and3-{2-[4-(1,1-dimethyl-propyl)-phenoxyl]acetylamino}-benzoic acid.Optionally, the number of codons between the nonsense mutation and thestart codon is selected from: 0, 1, 2, 3, 4, and 5. In certain case, thetarget protein is a secreted protein and the nonsense mutation isintroduced in a region of the nucleic acid which encodes a signalpeptide. Optionally, the nucleic acid is present in the genome of thecell or present on a vector (e.g., a viral vector).

In certain embodiments, the present invention provides a recombinantpolynucleotide molecule comprising a nucleic acid encoding a targetprotein, wherein the nucleic acid is operably linked to a promoter andhas been modified to contain at least one nonsense mutation downstreamto and in close proximity of the start codon that initiates translationof the target protein. Optionally, the number of codons between thenonsense mutation and the start codon is selected from: 0, 1, 2, 3, 4,and 5. In certain case, the target protein is a secreted protein and thenonsense mutation is introduced in a region of the nucleic acid whichencodes a signal peptide. Optionally, the nucleic acid is present in thegenome of the cell or present on a vector (e.g., a viral vector).

In certain embodiments, the present invention provides a host cellcomprising a polynucleotide molecule of the invention, such as apolynucleotide molecule comprising a nucleic acid encoding a targetprotein, wherein the nucleic acid is operably linked to a promoter andhas been modified to contain at least one nonsense mutation downstreamto and in close proximity of the start codon that initiates translationof the target protein. Preferably, the cell is a mammalian cell (e.g., ahuman cell). Optionally, the cell further comprises an agent whichsuppresses the nonsense mutation.

In certain embodiments, the present invention provides a transgenicnon-human animal, comprising a host cell of the invention. For example,the transgenic non-human animal is a mouse which contains a nucleic acidencoding a target protein, wherein the nucleic acid is operably linkedto a promoter and has been modified to contain at least one nonsensemutation downstream to and in close proximity of the start codon thatinitiates translation of the target protein.

In certain embodiments, the present invention provides a recombinantvector comprising: (a) a promoter; (b) a nucleic acid encoding a linkerpeptide, wherein the nucleic acid is operably linked to the promoter,and wherein the nucleic acid has been modified to contain at least onenonsense mutation located downstream to and in close proximity of thestart codon that initiates translation of the linker peptide; and (c) atleast one cloning site downstream to the nucleic acid for introducing atarget nucleic acid sequence which encodes a target protein to be fusedin frame to the carboxyl terminus of the linker peptide. For example,the linker peptide is a signal peptide (e.g., a native or a non-nativesignal peptide of the target protein).

In certain embodiments, the present invention provides a kit forregulating gene expression. The subject kit comprises a vectorcomprising: (a) a promoter; (b) an nucleic acid encoding a linkerpeptide, wherein the nucleic acid is operably linked to the promoter,and wherein the nucleic acid has been modified to contain at least onenonsense mutation located downstream to and in close proximity of thestart codon that initiates translation of the linker peptide; and (c) atleast one cloning site downstream to the nucleic acid for introducing atarget nucleic acid sequence which encodes a target protein to be fusedin frame to the carboxyl terminus of the linker peptide. For example,the linker peptide is a signal peptide (e.g., a native or a non-nativesignal peptide of the target protein). Optionally, the kit furthercomprises an agent that suppresses a nonsense mutation. Examples of theagent that suppresses the nonsense mutation include, but are not limitedto, an aminoglycoside antibiotic or an analog thereof, such as G418, andan acetylamino benzoic acid compound or a derivative thereof, such as3-[2-(4-tert-butyl-phenoxyl)-acetylamino]-benzoic acid and3-{2-[4-(1,1-dimethyl-propyl)-phenoxyl]acetylamino}-benzoic acid.

In certain embodiments, the present invention provides a method foridentifying a nucleic acid construct having a desired biologicalactivity from a population of nucleic acid constructs. In this method, apopulation of candidate nucleic acid constructs is produced, whereineach of the candidate nucleic acid construct is cloned into the multiplecloning sites of the vector which comprises: (a) a promoter; (b) annucleic acid encoding a linker peptide, wherein the nucleic acid isoperably linked to the promoter, and wherein the nucleic acid has beenmodified to contain at least one nonsense mutation located downstream toand in close proximity of the start codon that initiates translation ofthe linker peptide; and (c) at least one cloning site downstream to thenucleic acid for introducing a target nucleic acid sequence whichencodes a target protein to be fused in frame to the carboxyl terminusof the linker peptide. The population of candidate nucleic acidconstructs is expressed in a test cell. If a nucleic acid construct,when expressed in the test cell in the presence of an agent thatsuppresses the nonsense mutation, produces a desired biologicalactivity, then a nucleic acid construct having a desired biologicalactivity is identified from a population of nucleic acid constructs.

In certain embodiments, the present invention provides a method forproducing a transgenic nonhuman animal in which the expression of atarget protein can be modulated by an agent that suppresses a nonsensemutation. Such method comprises introducing a DNA construct into a germcell of a nonhuman animal or a germ cell of an ancestor of said animal,wherein said DNA construct comprises a nucleic acid encoding the targetprotein, wherein the nucleic acid is operably linked to a promoter andhas been modified to contain at least one nonsense mutation downstreamto and in close proximity of the start codon that initiates translationof the target protein. In certain embodiments, the present inventionprovides a transgenic non-human animal generated by such method.

In certain embodiments, the present invention provides a method forproducing a transgenic non-human animal in which the translation of atarget protein can be modulated by an agent that suppresses a nonsensemutation. Such method comprises: (a) providing a DNA molecule comprisinga nucleic acid sequence flanked at 5′ and 3′ ends by additionalpolynucleotide sequences of sufficient length for homologousrecombination between the DNA molecule and a chromosomal endogenoustarget region, wherein the nucleic acid has been modified to contain atleast one nonsense mutation downstream to and in close proximity of thestart codon that initiates translation of the target protein; (b)introducing the DNA molecule of (a) into a population of embryonic stemcells under conditions appropriate for homologous recombination betweenthe DNA molecule and the chromosomal endogenous target region to occur,resulting in embryonic stem cells in which the DNA molecule of (a) isintegrated into the chromosomal endogenous target region; (c) selectingan embryonic stem cell in which the DNA molecule of (a) is integrated atthe chromosomal endogenous target region; (d) implanting the embryonicstem cell selected from (c) into a blastocyst; and (e) implanting theblastocyst into a pseudopregnant foster mother, thereby producing anon-human transgenic animal in which the translation of the targetprotein can be modulated by an agent that suppresses the nonsensemutation. A preferred non-transgenic animal is a mammal (e.g., mouse).Optionally, the DNA molecule has integrated into one or two copies ofthe chromosomal endogenous target region. In certain embodiments, thepresent invention provides a transgenic non-human animal generated bysuch method.

In certain embodiments, the present invention provides a method forinducing expression of a target gene in a subject transgenic non-humananimal of the invention. Such method comprises administering to thetransgenic non-human animal an agent that suppresses the nonsensemutation. Examples of the agent that suppresses the nonsense mutationinclude, but are not limited to, an aminoglycoside antibiotic or ananalog thereof, such as G418, and an acetylamino benzoic acid compoundor a derivative thereof, such as3-[2-(4-tert-butyl-phenoxyl)-acetylamino]-benzoic acid and3-{2-[4-(1,1-dimethyl-propyl)-phenoxyl]acetylamino}-benzoic acid. Incertain cases, the agent is administered in a tissue-specific mannersuch that expression of the target gene is induced in a tissue-specificmanner. In other cases, the agent is administered in a temporal specificmanner such that expression of the target gene is induced in atemporal-specific manner.

In certain embodiments, the present invention provides a method foridentifying an agent that suppresses a nonsense mutation. Such methodcomprises (a) providing a test host cell which contains a polynucleotidemolecule comprising a nucleic acid encoding a target protein, whereinthe nucleic acid is operably linked to a promoter and has been modifiedto contain at least one nonsense mutation downstream to and in closeproximity of the start codon that initiates translation of the targetprotein; (b) providing a control host cell comprising a wild typenucleic acid encoding the target protein expressed under an appropriatepromoter and the expression of the target protein is at wild type level;(c) contacting the test host cell and the control host cell with acandidate agent; and (d) assaying for the level of the target protein inthe test host cell and in control host cell. If the candidate agentinduces translation of the target protein in the test host cell to asignificant level, the candidate agent is an agent that suppresses thenonsense mutation. Optionally, the target protein is fused to a reportermoiety and the expression of the reporter moiety is assayed. A preferredhost cell is a mammalian cell. In certain cases, the number of codonsbetween the nonsense mutation and the start codon is selected from; 0,1, 2, 3, 4 and 5. In certain specific embodiments, the target protein isa secreted protein, and the nonsense mutation is introduced in a regionof the nucleic acid which encodes a signal peptide.

In certain embodiments, the present invention provides a method foridentifying an agent that suppresses a nonsense mutation in vivo. Suchmethod comprises (a) providing a transgenic nonhuman animal whichcontains a polynucleotide molecule comprising a nucleic acid encoding atarget protein, wherein the nucleic acid is operably linked to apromoter and has been modified to contain at least one nonsense mutationdownstream to and in close proximity of the start codon that initiatestranslation of the target protein; (b) providing a control nonhumananimal which contains a wild type nucleic acid encoding the targetprotein expressed under an appropriate promoter and the expression ofthe target protein is at wild type level; (c) administering to thetransgenic animal and the control animal a candidate agent; and (d)assaying for the level of the target protein the transgenic animal andthe control animal. If the candidate agent induces translation of thetarget protein in the transgenic animal to a significant level, thecandidate agent is an agent that suppresses the nonsense mutation.Optionally, the target protein is fused to a reporter moiety and theexpression of the reporter moiety is assayed. A preferred host cell is amammalian cell. In certain cases, the number of codons between thenonsense mutation and the start codon is selected from: 0, 1, 2, 3, 4and 5. In certain specific embodiments, the target protein is a secretedprotein, and the nonsense mutation is introduced in a region of thenucleic acid which encodes a signal peptide.

In certain embodiments, the present invention provides a method ofinducing expression of a target gene in an individual. The method can becarried out, for example, by introducing cells containing a DNAconstruct described herein into an individual or by introducing a vectoror DNA construct described herein into an individual. In one embodiment,the method comprises (a) obtaining cells from the individual; (b)maintaining the cells under conditions appropriate for cell growth andcell division; (c) introducing into the cells of step (b) a DNAconstruct comprising a nucleic acid encoding a target protein, whereinthe nucleic acid is operably linked to a promoter and has been modifiedto contain at least one nonsense mutation downstream to and in closeproximity of the start codon that initiates translation of the targetprotein; (d) returning the cells produced in step (e) to the individual;and (e) administering to the individual an agent that suppresses thenonsense mutation. Alternatively, a DNA construct comprising a nucleicacid encoding a target protein, wherein the nucleic acid is operablylinked to a promoter and has been modified to contain at least onenonsense mutation downstream to and in close proximity of the startcodon that initiates translation of the target protein, can beintroduced into an individual in whom induction of expression of atarget gene is desired. The target protein to be induced by the methodcan be, for example, a therapeutic protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIGS. 1A-1C show design features of translation-based system for generegulation. FIG. 1A shows a schematic diagram of gene regulationstrategy. Relative positions of cap and polyadenylation sequences andinitiation and termination codons within mRNA are shown as well asproducts of translation in presence and absence of aminoglycoside. Brownstructure and green ‘ribbon’ denote ribosome translating mRNA andnascent polypeptides produced, respectively. FIG. 1B shows a sequenceconfiguration of initiation and termination codons used to generateinitial luc fusion proteins are shown, as well as structure oflentiviral vector used for studies. FIG. 1C shows levels of luc reportergene expression obtained with additional configurations of nonsensecodon sequences and adjacent sequences. Uninduced and induced refer tolevels of luc achieved in absence and presence of aminoglycoside; thevalues are expressed as the % of luc activity achieved in cells carryingthe luc construct possessing no nonsense mutations and not exposed todrug and represent the average of five independent determinations.Fold-induction refers to the ratio of induced to uninduced levels ofactivity.

FIGS. 2A-2C show data demonstrating aminoglycoside-induced suppressionof translational termination can be used to regulate gene expression incultured cells (panels A and B) Expression of luc in FG293 cellsinfected with lentiviral vectors encoding either the WT (panel A) ormutant (panel B) luciferase fusion product (see Results), as a functionof aminoglycoside concentration. Luc activity is measured in relativelight units (RLU); % of levels of luc activity achieved with WTluciferase fusion product are indicated for each drug concentration. (C)Kinetics of induction of luc gene expression in FG293 cells infectedwith the lentiviral vector encoding the mutant luc fusion product. Cellswere exposed to a constant concentration of G418 (200 μg/ml) and wereanalyzed for luc expression at hourly intervals. % WT luc activity isindicated for each time point, and represents the average of fiveindependent determinations.

FIG. 2D shows that aminoglycoside-induced suppression of translationaltermination can be used to regulate gene expression in three other celltypes (A549, B16, and C2C12).

FIG. 2E shows that aminoglycoside-induced suppression of translationaltermination results in induction of the reporter gene expression (about60%) as measured by fluorescence-activated cell sorting (FACS) analysis.The red line indicates induced cells, while the blue line indicatesuninduced cells.

FIG. 3A shows data demonstrating that Geneticin (G418) is the mosteffective suppressor of translation termination codons. Compounds weretested in vitro using FG293 cells infected with virus encoding themutant luc fusion product. Amount of expression achieved is expressed inrelative light units (RLU). Cells were exposed to drugs at the indicatedconcentrations for 48 hours prior to being assayed for luc expression,and the results represent the average of three independent experiments.

FIGS. 3B and 3C show that two novel compounds are effective insuppressing translation termination codons in FG293 cells. FIG. 3Bindicates induction (by3-[2-(4-tert-butyl-phenoxyl)-acetylamino]-benzoic acid. FIG. 3Cindicates induction by3-{2-[4-(1,1-dimethyl-propyl)-phenoxyl]acetylamino}-benzoic acid. Bothcompounds were purchased from Chembridge.

FIG. 4 shows that G418 effectively induces gene expression of humangrowth hormone gene (hGH) by suppressing translation termination codons,resulting in increased secretion of hGH protein as measured by ELISA.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, the present invention provides novelcompositions and methods of gene regulation that relies on the controlof translational termination. Applicants have shown that, using thenovel gene regulation system (e.g., compositions and methods) of theinvention, significant level of induction of expression of gene productscan be obtained. Such induction of expression is rapid, and regulationof expression can be achieved in a variety of in vitro and in vivocontexts. Applicants have also described several small molecule agentsthat effectively suppress nonsense mutation mediated translationaltermination in vitro and in vivo. The gene regulation system of thepresent invention is simple to implement, and allow the regulation ofgene expression to occur in the context of the normal endogenous controlelements of a target gene. Certain examples of the target genes includea mammalian gene which encodes a secreted protein.

For example, the gene regulation system of the present invention relatesto a method for inducing expression of a target protein in a host cell.Such method comprises contacting the host cell with an effective amountof an agent, wherein the host cell comprises a nucleic acid encoding atarget protein, wherein the nucleic acid is operably linked to apromoter and has been modified to contain at least one nonsense mutationdownstream to and in close proximity of the start codon that initiatestranslation of the target protein, and wherein the agent suppresses thenonsense mutation.

The term “expression,” as used herein, refers to the biological processof producing an active polypeptide from the nucleic acid molecule orgene that encodes it. The term is intended to encompass some or all ofthe steps of normal genetic expression including, transcriptionalinitiation, transcription, transcript processing, translation, andpost-translational processing.

The term “nonsense mutation” or “stop codon mutation,” as used herein,is intended to refer to a mutation that results in the introduction of anon-naturally-occurring stop codon in a coding region of a targetnucleic acid. The term “coding region” refers to nucleotide sequence ofa gene which specifies the curious for translation into proteins, e.g.,the region that codes for the amino acids. Coding region sequences areto be distinguished from other non-coding sequences, for example, 5′untranslated regions, 3′ untranslated regions, and introns.

The term “suppress a nonsense mutation,” as used herein, is intended toencompass counteracting or overcoming, the effect of the nonsensemutation, either partially or entirely, such that translation of atarget protein from its encoding nucleic acid is partially or fullyrestored. As a result, expression of a target protein is partially orfully restored.

Nonsense Mutations and Nucleic Acid Molecules Comprising NonsenseMutations

In certain embodiments, the present invention relates to a recombinantpolynucleotide molecule comprising a nucleic acid encoding a targetprotein, wherein the nucleic acid is operably linked to a promoter andhas been modified to contain at least one nonsense mutation downstreamto and in close proximity of the start codon that initiates translationof the target protein. The term “modified” refers to the deliberatemodification of a coding region of a target nucleic acid (e.g., a geneor a portion thereof) and is not intended to encompass naturallyoccurring nonsense mutations that may exist in the a target nucleicacid. In certain case, the polynucleotide molecule is present in thegenuine of a cell. Alternatively, the polynucleotide molecule is presenton a vector (e.g., a viral vector).

Optionally, the nonsense mutation is positioned close to the beginningof the target protein coding sequence. To illustrate, the number ofcodons between the nonsense mutation and the start codon is selectedfrom: 0, 1, 2, 3, 4, and 5. In some cases, the nonsense mutation isintroduced into a target nucleic acid with some additional sequencesflanking the nonsense mutation. For example, a naturally occurringnonsense mutation in the human Apo CII gene (see, e.g., FIG. 1B) can beintroduced into a target nucleic acid. Alternatively, artificiallydesigned nonsense mutation-containing nucleotide sequence may beintroduced into a target nucleic acid. Optionally, a nonsense mutationis positioned in a target nucleic acid in such a way that an mRNAcontaining the nonsense mutation is not subject to nonsense-mediatedmRNA decay (NMD).

In certain specific embodiments, the present invention relates toregulation of expression of a target protein which is a secretedprotein. For example, the nonsense mutation is introduced in a region ofthe nucleic acid which encodes a signal peptide of the secreted proteinsuch that the structure of the mature protein is not affected by theintroduction of the nonsense mutation.

In certain specific embodiments, the present invention provides arecombinant vector comprising: (a) a promoter; (b) an nucleic acidsequence encoding a linker peptide, wherein the nucleic acid is operablylinked to the promoter, and wherein the nucleic acid has been modifiedto contain at least one nonsense mutation located downstream to and inclose proximity of the start codon that initiates translation of thelinker peptide; and (c) at least one cloning site downstream to thenucleic acid for introducing a target nucleic acid sequence whichencodes a target protein to be fused in frame to the carboxyl terminusof the linker peptide. As used herein, a “linker peptide” refers to apeptide that is fused at the carboxyl terminus of a target protein anddoes not contribute to the functions of the target protein. Preferably,the linker peptide can be readily removed from the target protein suchas by enzyme digestion or post-translational cleavage. Examples of suchlinker peptide include, but are not limited to, signal peptides andlabeling peptides (e.g., histidine tag, Myc, Fc, GST). When the linkerpeptide is a signal peptide, it is understood that the signal peptidecan be a native or a non-native signal peptide of the target protein.

It is known in that art that translation is almost always initiated at amethionine codon (AUG) codon, and the end of translation is signaled byone of three possible stop codons (UAG, UAA, UGA). In the presentinvention, expression of a target protein is perturbed by introducing atleast one nonsense mutation (e.g., UAG, UAA, UGA) into a coding sequenceto perturb normal translation of the target protein. For example, one ofthe three stop codons (i.e., UAG, UGA, UAA) is created from a single,double, or triple nucleotide mutation in the nucleotides of one or moreselected amino acid-specifying codons. Genes that have such mutationsintroduced into their coding region, upon translation, produce geneproducts that are terminated at the codon of the mutation. Optionally,the nonsense mutation is introduced in close proximity of the startcodon, resulting in no expression of the target protein or a truncatedinactive version of the target protein. Preferably, introduction of thenonsense mutation does not result in alterations of the kinetics ofmessenger RNA stability.

Methods of introducing a nonsense mutation in a target nucleic acid areknown in the art. Some or all of the nucleotide sequence of the codingregion of the desired gene must be known to allow for design of theappropriate introduced mutation(s). For desired genes in which thesequence of some or all of the coding region is not known, standard DNAsequencing methods can be used to obtain the sequence of some or all ofthe coding region of the gene.

For example, the nonsense mutations of the invention are introduced intoa desired gene through methods of directed mutagenesis. As used herein,the term “directed mutagenesis” (also referred to as site-directedmutagenesis) encompasses methods of introducing a specific andpredetermined mutation into a known nucleotide sequence. Furthermore, asused herein, the term “directed mutagenesis” implies that some or all ofthe nucleotide sequence that is intended to be mutated has beendetermined or is known. For example, one or more specific mutations maybe introduced into the coding region of a desired gene by one skilled inthe art using directed mutagenesis methods involving specificallydesigned oligonucleotides, e.g., oligonucleotide-directed mutagenesis.These methods typically involve the introduction of the desired geneinto a vector that can be reliably replicated from an oligonucleotideprimer. Oligonucleotide primers with one or more nucleotide mismatchesinside the hybridizing region (i.e., the region that hybridizes to thedesired gene) can be readily designed and used by one skilled in the artto introduce mutations of the invention into a desired gene by standardrecombinant DNA techniques known in the art.

In certain specific embodiments, a viral vector replication system isused for oligonucleotide-directed mutagenesis, e.g., an M13 viralvector. Methods for introducing mutations of the invention with M13 andother viral vectors are readily available in the art (see, for example,Kunkel, 1985, PNAS U.S.A. 82:488-492; Kunkel et al, 1987, Meth.Enzymol., 154;367-382). In other specific embodiments, a bacterialplasmid replication system is used, in which the desired gene isintroduced into the bacterial plasmid and the mutation(s) of theinvention is deliberately introduced into the desired gene witholigonucleotide primers containing one or more mismatches at preselectednucleotide positions. Several such systems have been described that canbe readily used by one skilled in the art (see, for example, Deng andNikoloff, 1992, Anal. Biochem. 200:81-88; Nikoloff et al, 1996,58:455-468).

In certain embodiments of the invention, mutations of the invention maybe introduced into desired genes during amplification with PCR(polymerase chain reactions), also referred to as PCR-mediatedmutagenesis. A number of PCR-based methods to deliberately introducemutations into a desired gene are available in the art and can be usedin the invention (see, for example, Ausubel et al, Current Protocols inMolecular Biology, John Wiley & Sons, 1995). These methods generallyinvolve the use of oligonucleotide primers for PCR amplificationreactions that contain one or more nucleotide mismatches within thehybridizing region (i.e., the region of hybridization with the desiredgene).

In certain embodiments of the invention, mutations of the invention maybe introduced into a desired gene through methods of non-directedmutagenesis. As used herein, the term “non-directed mutagenesis” refersto any method of introducing mutations randomly into the desired gene ina replicable biological system and having a means of screening resultantnucleic acids and to select and use the one(s) which contains a mutationof the invention in a desired gene. Such non-directed mutagenesis can beaccomplished through a variety of means, the example, by exposure of thegene and/or the replicable biological system in which it is propagated,to mutagenic conditions, e.g., irradiation, chemical mutagens and thelike. Other methods available to one skilled in the art include anymethod involving enzymatic replication of nucleic acid, for example witha polymerase or a replicas, whereby the intrinsic replication error rateof the enzyme is able to accomplish non-directed mutagenesis. As usedherein, “screening” of the resultant nucleic acids to find a desiredgone with a mutation of the invention includes methods for determiningnucleotide sequences, e.g., sequencing analysis. In addition, the term“screening” also includes methods for determining amino acid sequencesof protein, e.g. microsequencing. The term “screening” furthermoreincludes biological assays that can discern mutations of the inventionin a desired gene, in particular mutations that result in the productionof an inactive (partially or completely) gene product upon translationof the desired gene. Depending on which desired gene is selected, theordinarily skilled artisan can select an appropriate known biologicalassay by which to screen randomly generated mutants to thereby selectmutants that result in the production of an inactive (partially orcompletely) gene product upon translation of the desired gene.

In still another embodiment, a mutation of the invention is introducedinto a desired gene that is present within the genome of a eukaryoticcell (i.e., an endogenous gene) by manipulation o the endogenous genesequence, preferably by homologous recombination. Methods of usinghomologous recombination to alter the sequence of an endogenouseukaryotic gene are well known in the art and can be applied todeliberately introduce one or more mutations or the invention into adesired endogenous eukaryotic gene. Such methods typically involveconstruction of a gene targeting vector comprising at least a portion ofthe endogenous gene sequence, introduction of the targeting vector intoa eukaryotic cell containing the endogenous desired gene and cultureunder conditions sufficient to allow for homologous recombinationbetween the targeting vector and the endogenous gene, followed byscreening of clones for ones in which the endogenous gene has undergonehomologous recombination with the targeting vector using standardrecombinant DNA techniques. To introduce one or more mutations into theendogenous gene, the targeting vector includes at least a portion of theendogenous gene sequence into which the mutation(s) has been introduced,flanked by wildtype sequences of the endogenous gene to facilitatehomologous recombination between the targeting vector and the endogenousgene.

The Examples below further describe in detail how to introduce nonsensemutations into various target nucleotide sequences including humangrowth hormone (hGH) gene and how to design and select appropriatenonsense mutations that can be used for effectively inducing expressionof a target protein in the presence of an agent that suppresses thenonsense mutation.

Vectors, Cell Lines, and Transgenic Animals

In certain embodiments, the present invention relates to methods ofinserting at least one nonsense mutation into an endogenous target genein a cell, or into an exogenous target gene to be introduced into a cellby a vector. In either case, necessary elements (e.g., promoters) arepresent for the transcription of the modified target gene which containsthe nonsense mutation. For example, such modified target gene is placedinto an appropriate expression vector which contains the necessaryelements for the transcription of the modified target gene. Theexpression vector is then transfected into a host cell in order toeffectuate expression of the modified target gene in the absence or inthe presence of an agent that suppresses the nonsense mutation. In afurther embodiment, the expression vector, which contains the necessaryelements for the transcription of the modified target gene, isintroduced into an individual (without having first been introduced intoa host cell that is, in turn, introduced into an individual).

Vectors comprising a modified target gene which contains a nonsensemutation according to the present invention can be manufacturedaccording to methods generally known in the art, for example, bychemical synthesis or recombinant DNA/RNA technology (see, e.g.,Sambrook et al., Eds., Molecular Cloning: A Laboratory Manual, 2ndedition, Cold Spring: Harbor University Press, New York (1989); andAusubel et al., Eds., Current Protocols in Molecular Biology, John Wiley& Sons, New York (1997).

In certain embodiments, the present invention provides a kit forregulating gene expression. The subject kit comprises a vector whichcomprises: (a) a promoter; (b) an nucleic acid encoding a linkerpeptide, wherein the nucleic acid is operably linked to the promoter,and wherein the nucleic acid has been modified to contain at least onenonsense mutation located downstream to and in close proximity of thestart codon that initiates translation of the linker peptide; and (c) atleast one cloning site downstream to the nucleic acid for introducing atarget nucleic acid sequence which encodes a target protein to be fusedin frame to the carboxyl terminus of the linker peptide. For example,the linker peptide is a signal peptide, which may be a native or anon-native signal peptide of the target protein. Optionally, the kit mayfurther comprise an agent that suppresses a nonsense mutation. Toillustrate, such agent is an aminoglycoside antibiotic or an analogthereof (e.g., G418). In certain cases, the agent is an acetylaminobenzoic acid compound or a derivative thereof, such as3-[2-(4-tert-butyl-phenoxyl)-acetylamino]-benzoic acid or3-{2-[4(1,1-dimethyl-propyl)-phenoxyl]acetylamino}-benzoic acid.

In certain embodiments, the subject vector or nucleic acid of thepresent invention comprises a nucleotide element sufficient forinitiation of transcription (such as a promoter) operably linked to thenucleic acid encoding the desired target protein. Examples of promotersinclude, but are not limited to, tRNA promoter, 5S rRNA promoters,histone gene promoters, CMV promoter, RSV promoter, SV40 promoter, PEPCKpromoter, MT promoter, SRα promoter, P450 family promoters, GAL7promoter, T₇ promoter, T₃ promoter, SP6 promoter, and K11 promoter. TheT7 promoter, T₃ promoter, SP6 promoter, and K₁₁ promoter have beendescribed U.S. Pat. No. 5,591,601, the entire contents of which areincorporated by reference.

In certain embodiments, the invention relates to packaging cell linesuseful for generating recombinant viral vectors and viruses comprising arecombinant genome which includes a nucleotide sequence (RNA or DNA)which represents a vector or nucleic acid of the present invention;construction of such cell lines; and methods of using, the recombinantviral vectors to modulate production of a desired target protein invitro, in vivo and ex vivo. In a particular embodiment, the recombinantviral vectors and viruses comprise a recombinant genome which includes anucleic acid encoding a target protein, wherein the nucleic acid isoperably linked to a promoter and has been modified to contain at leastone nonsense mutation downstream to and in close proximity of the startcodon that initiates translation of the target protein. Cell linesuseful fur generating recombinant viral vectors and viruses are producedby transfecting host cells, such as mammalian host a viral vectorincluding the subject nucleic acid integrated into the genome of thevirus, as described herein. Viral stocks are harvested according tomethods generally known in the art. See, e.g., Ausubel et al Eds.,Current Protocols In Molecular Biology, John Wiley & Sons, New York(1998); Sambrook et al., Eds., Molecular Cloning; A Laboratory Manual,2nd edition, Cold Spring Harbor University Press, New York (1989); Danosand Mulligan, U.S. Pat. No. 5,449,614; and Mulligan and Wilson, U.S.Pat. No. 5,460,959, the teachings of which are incorporated herein byreference. The recombinant viral vectors produced by the packaging, celllines of the present invention are also referred to herein as viralvectors which represent the subject nucleic acid.

In certain embodiments, the present invention relates to cells (hostcells) which comprise a subject nucleic acid of the invention (e.g., amodified target gene which contains the nonsense mutation). Particularcells which comprise a DNA construct of the invention are discussedabove.

In a particular embodiment, a subject nucleic acid construct of theinvention can be used to produce transgenic animals whose cells containand express the nucleic acid construct (e.g., a modified target genewhich contains the nonsense mutation). There is a variety of techniquesfor producing transgenic animals of the present invention. For example,foreign nucleic add can be introduced into the germline of an animal by,for example, introducing the additional foreign genetic material into agamete, such as an egg. Alternatively, transgenic animals can beproduced by breeding animals which transfer the foreign DNA to theirprogeny. It is also possible to produce transgenic animals byintroducing foreign DNA into somatic bells from which an animal isproduced. As used herein, the term “transgenic animal” includes animalsproduced from cells modified to contain foreign DNA or by breeding; thatis, it includes the progeny of animals (ancestors) which were producedfrom such modified cells. As used herein, the term “foreign nucleicacid” refers to a genetic material obtained from a source other than theparental germplasm. Preferably, the transgenic animals are derived frommammalian embryos.

In certain aspects, the invention provides a homologous recombinantnon-human animal expressing a subject nucleic acid construct of theinvention. The term “homologous recombinant animal” as used herein isintended to describe an animal containing a gene which has been modifiedby homologous recombination between the gene and a DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal. For example, an animal can be created in which a target genewhich has been modified to contain a nonsense mutation is introducedinto a specific site of the genome.

In certain cases, to create such a homologous recombinant animal, avector is prepared which contains a nonsense mutation flanked at its 5′and 3′ ends by additional nucleic acid of a eukaryotic gene at whichhomologous recombination is to occur. The additional nucleic acidflanking the nonsense mutation is of sufficient length for successfulhomologous recombination with the eukaryotic gene. Typically, severalkilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector (see e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell51:503 for a description of homologous recombination vectors). Thevector is introduced into an embryonic stein cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected (see e.g., Li, E, et al.(1992) Cell 69:915).

In addition to the homologous recombination approaches described above,enzyme-assisted site-specific integration systems are known in the artand can be applied to the components of the regulatory system of theinvention to Integrate a DNA molecule at a predetermined location in asecond target DNA molecule. Examples of such enzyme-assisted integrationsystems include the Cre recombinase-lox target system (e.g., asdescribed in Baubonis, W, and Sauer, B. (1993) Nucl. Acids Res.91:2025-2029; and Fukushige, S. and Sauer, B. (1992) Proc. Natl. Acad.Sci. USA 89;7905-7909) and the FLP recombinase-FRT target system (e.g.,as described in Dang, D. T. and Perrimon, N. (1992) Dev. Genet.13:367-375; and Fiering, S. et al. (1993) Proc. Natl. Acad. Sci. USA90:8469-8473).

Methods for acquiring, culturing, maintaining and introducing foreignnucleic acid sequences into recipient eggs for transgenic animalproduction are well known in the art. See, for example, Manipulating theMouse Embryo: A Laboratory Manual, Hogan et al., Cold Spring HarborLaboratory, New York (1986). Preferably, a DNA construct will bedelivered into the embryo at a very early stage in development so thatonly a small frequency of the embryos are mosaic (e.g., an embryo inwhich integration of the foreign nucleic acid occurs after the one cellstage of development).

Methods of Delivering Nucleic Acids

In certain embodiments, the subject nucleic acid (e.g., a modifiedtarget gene which contains the nonsense mutation) can be introduced intoa cell by a variety of methods (e.g., transformation, transfection,direct uptake, projectile bombardment, using liposomes). The presentinvention contemplates any methods generally known in the art which areappropriate for the particular agent or effector and cell type. Forexample, agents and effectors can be introduced into a cell by directuptake, DEAE-dextran, calcium phosphate precipitation, lipofection, cellfusion, electroporation, biolistics, microinjection, infection (e.g., byDNA viruses and RNA viruses) and retrovirus-mediated transduction. Suchmethods are described in more detail, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor University Press, New York (1989); and Ausubel, et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. (1998).Other suitable methods are also described in the art.

For example, a subject vector of the present invention can also beintroduced into a cell by targeting the vector to cell membranephospholipids. For example, targeting of a vector of the presentinvention can be accomplished by linking the vector molecule to a VSV-Gprotein, a viral protein with affinity for all cell membranephospholipids. Such a construct can be produced using methods well knownto those practiced in the art. In those embodiments of the method inwhich a vector that contains the necessary elements for thetranscription of the modified target gene is introduced into anindividual, the vector can be introduced by any route which results indelivery to the desired location(s) in the individual. For example, itcan be injected (e.g., intramuscularly, intraperitoneally,subcutaneously); introduced intravenously, rectally, orally, or byinhalation; applied topically/administered transdermally or by any otherappropriate parenteral or nonparenteral route. In these embodiments, thevector can be present in an appropriate carrier, such as water,physiological buffer, or a lipid based carrier. Other routes ofadministration and carriers are described herein.

In a particular embodiment, vectors of the invention include, but arenot limited to, a DNA plasmid, virus or other suitable replicon (e.g.,viral vector). Viral vectors include retrovirus, adenovirus, parvovirus(e.g., adeno-associated viruses), coronavirus, negative strand RNAviruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus(e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.,measles and Sendai), positive strand RNA viruses such as picornavirusand alphavirus, and double stranded DNA viruses including adenovirus,herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox andcanarypox). Other viruses include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include: avian leukosis-sarcoma, mammalianC-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus,spumavirus (Coffin, J. M., Retroviridae: The viruses and theirreplication, In Fundamental Virology, Third Edition, B. N. Fields, etal., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Otherexamples include murine leukemia viruses, murine sarcoma viruses, mousemammary tumor virus, bovine leukemia virus, feline leukemia virus,feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus,baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkeyvirus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcomavirus and lentiviruses. Other examples of vectors are described, forexample, in McVey et al., U.S. Pat. No. 5,801,030, the teachings ofwhich are incorporated herein by reference.

Virus stocks consisting of recombinant viral vectors comprising arecombinant genome which includes a nucleic acid of the presentinvention (e.g., a modified target gene which contains the nonsensemutation), are produced by maintaining the transfected cells underconditions suitable for virus production. Such conditions, which are notcritical to the invention, are generally known in the art. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor University Press, New York (1989); Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York(1998); U.S. Pat. No. 5,449,614; and U.S. Pat. No. 5,460,959, theteachings of which are incorporated herein by reference. The resultingrecombinant viral vectors can be used, as described herein, to modulateproduction of a desired target protein in vitro, in vivo and ex vivo.

Regulation of Gene Expression

In certain embodiments, the present invention relates to methods ofregulating expression of a target protein in vitro, in vivo, and exvivo, after one or more nonsense mutations have been introduced into anucleic acid encoding the target protein. Expression of the targetprotein is regulated by contacting cell with or administering to ananimal an agent that suppresses the nonsense mutation, such thatexpression of the target protein is induced in the cell or animal to asignificant level relative to an appropriate control cell or animal. TheExamples below describe in detail how to determine a significant levelof induction relative to an appropriate control cell or animal.

For example, in certain cases, “a significant level” of induction can berepresented by a number of fold of induction by a nonsense mutationsuppressor. To illustrate, “a significant lever” of induction may berepresented by a 20-, 40-, 60-, 100-, or 200-fold of induction mediatedby a nonsense mutation suppressor. In these cases, an “appropriatecontrol” cell or animal refers to a cell or animal in which an agentwhich suppresses the nonsense mutation is not contacted or administered,while the control cell or animal, like the test cell or animal,comprises a nucleic acid construct (e.g., a nucleic acid which has beenmodified to contain one or more nonsense mutations).

In other cases, “a significant level” of induction, as used herein, canbe represented by a high level of suppression of translation by anonsense mutation suppressor. In this case, the level of suppression oftranslation is calculated as a level of the target protein produced by amodified nucleic acid which contains a nonsense mutation, relative to alevel of the target protein produced by a wildtype nucleic acid whichdoes not contain the nonsense mutation. To illustrate, “a significantlevel” of induction is achieved if a target protein is produced from amodified nucleic acid in the presence of a nonsense mutation suppressorat 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the expression levelproduced from a wildtype nucleic acid. In these cases, an “appropriatecontrol” cell or animal refers to a cell or animal which comprises awildtype nucleic acid construct (e.g., a nucleic acid does not contain anonsense mutation).

In one embodiment, an agent that suppresses the nonsense mutation (alsoreferred to as a suppressor or inhibitor of nonsense mutation) is anaminoglycoside. Aminoglycosides are a class of compounds with antibioticproperties. It is known that at least some of their antibiotic effectoccurs through a mechanism whereby translation at the ribosomal subunitsis disrupted. Aminoglycosides preferentially bind to the 30S eukaryoticribosomal subunit. At certain subtoxic concentrations, the partialeffect of aminoglycosides on the translation machinery is to reduce thefidelity of the translation machinery, such that codons for terminationare misread to be codons for amino acids (e.g., tyrosine). Examples ofthe aminoglycosides include, but are not limited to, hygromycin-B,paromomycin, tobramycin, gentamycin, and G418. As used herein, the term“aminoglycoside” is intended to include pharmaceutically acceptablesalts thereof, such as sulfates thereof (e.g., gentamycin sulfate).

In another embodiment, an agent that suppresses the nonsense mutation isan acetylamino benzoic acid compound or a derivative thereof. Examplesof the acetylamino benzoic acid compound includes, but are not limitedto, 3-[2-(4-tert-butyl-phenoxyl)-acetylamino]-benzoic acid and3-{2-[4-(1,1-dimethyl-propyl)-phenoxyl]acetylamino}-benzoic acid.Further exemplary acetylamino benzoic acid compounds or derivativesthereof are described in the art (see, e.g., published PCT applicationWO 2004/009533, the teachings of which are incorporated herein byreference).

In certain embodiments, the present invention relates to regulatingexpression of a target protein which has therapeutic applications (alsoreferred to as a “therapeutic protein”). Examples of the therapeuticproteins include, but are not limited to, erythropoietin, insulin,vascular endothelial cell growth factors (VEGFs); modified VEGF receptoror fragments thereof, fibroblast growth factors (FGFs), Hypoxia-InducingFactor-1α (HTF-1α), Factor VIII, Factor IX, Growth Hormone, endostatin,angiostatin and Herpes Simplex Virus Thymidine Kinase.

Screening Methods

In certain embodiments, the present invention relates to methods forscreening for suppressors of a nonsense mutation which can induceexpression of the target protein in a cell or an animal to a significantlevel relative to an appropriate control cell or animal. Optionally,such an agent may be able to suppress all three nonsense mutations.Alternatively, such an agent may be able to suppress a specific nonsensemutation, and not other nonsense mutations. A suppressor of a nonsensemutation is utilized in the gene regulation methods of the invention ata concentration that effectively suppresses the nonsense mutation in thetarget gene, but is not toxic to the host cell.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. Agents to be testedfor their ability to suppress nonsense mutations can be produced, forexample, by bacteria, yeast, plants or other organisms (e.g., naturalproducts), produced chemically (e.g., small molecules), or producedrecombinantly. Candidate agents contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. Incertain embodiments, the candidate agent is an antibiotic. In otherembodiments, the candidate agent is an acetylamino benzoic acidcompound. In certain further embodiments, the candidate agent is ananalogue or derivatives of aminoglycoside antibiotics or acetylaminobenzoic acid compounds.

The test agents cart be provided as single, discrete entities, orprovided in libraries of greater complexity, such as made bycombinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amities, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps.

In some cases, one or more compounds can be tested simultaneously. Wherea mixture of compounds is tested, the compounds selected by theforegoing processes can be separated (as appropriate) and identified bysuitable methods (e.g., PCR, sequencing, chromatography). Largecombinatorial libraries of compounds (e.g., organic compounds, peptides,nucleic acids) produced by combinatorial chemical synthesis or othermethods can be tested (see e.g., Ohlmeyer, M. H. J. et al., Proc. Natl.Aced. Sci. USA 90:10922-10926 (1993) and DeWitt, S. H. et al., Proc.Natl. Acad. Sci. USA 90:6909-6913 (1993), relating to tagged compounds;see also, Rutter, W. J. et al., U.S. Pat. No. 5,010,175; Huebner, V. D.et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No.4,833,092).

Methods of Treatment and Administration

In certain embodiments, agents and effectors of the present invention,including a subject nucleic acid (e.g., a modified nucleic acid encodinga target protein) and a suppressor of a nonsense mutation, can beintroduced into a cell for therapeutic applications. As used herein, acell includes, but is not limited to, a eukaryotic cell, such as ananimal, plant or yeast cell. A cell which is of animal or plant origincan be a stem cell or somatic cell. Suitable animal cells can be of, forexample, mammalian or avian origin. Examples of mammalian cells includehuman (such as HeLa cells), bovine, ovine, porcine, marine, (such asembryonic stem cells), rabbit and monkey (such as COS1 cells) cells. Thecell may be an embryonic cell, bone marrow stem cell or other progenitorcell. Where the cell is a somatic cell, the cell can be, for example, anepithelial cell, fibroblast, smooth muscle cell, blood cell (including ahematopoietic cell, red blood cell, T-cell, B-cell, etc.), tumor cell,cardiac muscle cell, macrophage, dendritic cell, neuronal cell (e.g., aglial cell or astrocyte), or pathogen-infected cell (e.g., thoseinfected by bacteria, viruses, virusoids, parasites, or prions).

The cells can be obtained commercially or from a depository or obtaineddirectly from an individual, such as by biopsy. The cells used can beobtained from an individual to whom they will be returned or fromanother/different individual of the same or different species. Forexample, nonhuman cells, such as pig cells, can be modified to include aDNA construct and then introduced into a human. Alternatively, the cellneed not be isolated from the individual where, for example, it isdesirable to deliver the vector to the individual in gene therapy.

For example, the present invention relates to a method of regulatingexpression of an endogenous gene (a gene resident in a cell as the cellwas obtained) to produce a desired target protein and compositionsuseful in the method. The endogenous gene can be one which is expressed(“on”) in the cell or one which is normally not expressed (“off”) in thecell but whose expression is or has been turned on or activated. Forexample, a nucleic acid which has been modified to contain a nonsensemutation, or a virus or viral vector comprising such nucleic acid, canbe introduced into genomic DNA of cells. As a result of the introductionof the nonsense mutation, a target protein encoded by the nucleic acidis not produced or produced at a low level in the cells in the absenceof a suppressor of the nonsense mutation. In the presence of asuppressor of the nonsense mutation, translation of the target proteinis restored, leading to expression of the target protein. In analternative embodiment, a nucleic acid which has been modified tocontain a nonsense mutation, or a virus or viral vector comprising suchnucleic acid, is introduced into the endogenous gene encoding the targetprotein. The resulting cells can be used, as described herein, toregulate production of the target protein in an individual.

The nucleic acids of the present invention can be used in methods ofinducing expression of a target protein in an individual (e.g., a humanor other mammal or vertebrate). In these methods, a nucleic acid of thepresent invention can be introduced into cells obtained from theindividual. The cells can be migratory, such as a hematopoietic cell, ornon-migratory, such as a solid tumor cell or fibroblast. After treatmentin this manner, the resulting cells can be administered to (introducedinto) the individual according to methods known to those practiced inthe art. To induce expression of the target protein, a nonsense mutationsuppressor can be administered to the individual according to methodsknown to those practiced in the art. Such a treating procedure issometimes referred to as ex vivo treatment. Ex vivo therapy has beendescribed, for example, in Kasid, et al., Proc. Natl. Acad. Sci. USA,87:473 (1990); Rosenberg et al., N. Engl. J. Med., 323:570 (1990);Williams et al., Nature, 310:476 (1984); Dick et al., Cell, 42:71(1985); Keller et al., Nature, 318:149 (1985); and Anderson et al., U.S.Pat. No. 5,399,346.

In certain particular embodiments, the nucleic acids or vectors of thepresent invention can be administered directly to the individual inorder to express (induce expression of) a target protein in anindividual. The mode of administration is preferably at the location ofthe target cells. The administration can be nasally or by injection.Other modes of administration (parenteral, mucosal, systemic, implant,intraperitoneal, oral, intradermal, transdermal, intramuscular,intravenous including infusion and/or bolus injection, subcutaneous,topical, epidural, buccal, rectal, vaginal, etc.) are generally known inthe art. The nucleic acids can, preferably, be administered in apharmaceutically acceptable carrier, such as saline, sterile water,Ringer's solution, or isotonic sodium chloride solution. A nonsensemutation suppressor is then administered to the individual, in whomtranslation of the target protein is restored, resulting in productionof the target protein.

In certain specific embodiments, suppressors of a nonsense mutation areaminoglyosides. Aminoglycosides have been used both in vitro and in vivofor antibiotic purposes and, in certain limited situations, to suppressnaturally-occurring stop codon mutations. Thus dosages and routes ofadministration of aminoglycosides that have been used to suppressnaturally-occurring stop codon mutations also can be used to suppressthe deliberate mutations of the invention. For example, a non-limitingdosage range of aminoglycoside for use with mammalian cells in cultureis 0.05 mg/ml to 1 mg/ml, preferably 0.2 mg/ml. Aminoglycosides arecommercially available (e.g., from Sigma Chemical Company) and can beadded to culture medium in an aqueous solution. For in vivo treatment,pharmacokinetic data generated in the dog (Morris, T. H. (1995) Lab.Animal 29:16-36) can be used to calculate the allometric dose equivalentfor other species of subjects (e.g., humans). For example, for mice aneffective dosage for gentamycin sulfate has been found to be 17 mg/kg(Barton-Davis, E. R. (1999) J. Clin. Invest, 104:375-381). Theaminoglycoside can be delivered by injection in vivo, such as bysubcutaneous injection. Sustained treatment with the aminoglycoside maybe necessary in vivo to maintain sustained expression of the gene ofinterest. For example, in one embodiment, the aminoglycoside isadministered by subcutaneous injection at 100%, 200% and 400% of thecalculated dose equivalents (based on Morris, T. H. supra) once per dayfor 14 days. In another embodiment, the aminoglycoside is administeredusing an osmotic pump. The osmotic pump can be implanted under the skinof the subject. In one embodiment, the pump is loaded with appropriatedrug concentrations for the subject to receive 50%, 100% or 200% of thecalculated dosage for two weeks.

In other specific embodiments, the present invention relates toacetylamino benzoic acid compounds or derivatives thereof as suppressorsof a nonsense mutation. Like aminoglycosides, when these suppressors areto be used in vivo in a subject, a particular compound is chosen that issuitable for in vivo use (e.g., a compound in which any potential sideeffects are not so severe as to preclude use in vivo). Preferred dosageranges, and potential toxicity, of these suppressor compounds can bedetermined using in vitro systems (e.g., cultured cells) or animal modelsystems. Moreover, the dosage of suppressor compounds to be used eitherin vitro or in vivo may be adjusted over time to thereby adjust thelevel of expression of the desired gene.

In certain specific embodiments, agents and effectors can beadministered to an individual in a variety of ways. The route ofadministration depends upon the particular agent or effector. Routes ofadministration are generally known in the art and include oral,intradermal, transdermal (e.g., in slow release polymers),intramuscular, intraperitoneal, intravenous including infusion and/orbolus injection, subcutaneous, topical, epidural, buccal, rectal,vaginal and intranasal routes. Other suitable routes of administrationcan also be used, for example, to achieve absorption through epithelialor mucocutaneous linings.

The dosage of agent and effector of the present invention administeredto an individual, including frequency of administration, will varydepending upon a variety of factors, including mode and route ofadministration; size, age, sex, health, body weight and diet of therecipient; nature and extent of symptoms of the disease or disorderbeing treated; kind of concurrent treatment, frequency of treatment, andthe effect desired.

Pharmaceutical Compositions

In certain embodiments, the agent and effector of the presentdisclosure, including a subject nucleic acid (e.g., a modified nucleicacid encoding a target protein) and a nonsense mutation suppressor,collectively referred to herein as therapeutic agents, are formulatedwith a pharmaceutically acceptable carrier. Such therapeutic agents canbe administered alone or as a component of a pharmaceutical formulation(composition). The therapeutic compositions of the invention can be usedalone or in admixture, or in chemical combination, with one or morematerials, including other recombinant vectors, materials that increasethe biological stability of the recombinant vectors, or materials thatincrease the ability of the therapeutic compositions to specificallypenetrate the relevant cell type. The therapeutic compositions of theinvention are administered in pharmaceutically acceptable carriers(e.g., physiological saline), which are selected on the basis of themode and route of administration, and standard pharmaceutical practice.Suitable pharmaceutical carriers, as well as pharmaceutical necessitiesfor use in pharmaceutical formulations, are described in Remington'sPharmaceutical Sciences, a standard reference text in this field.

The therapeutic compositions of the invention are administered indosages determined to be appropriate by one skilled in the art. Anappropriate dosage is one that effects a desired result, e.g., areduction in a symptom of a disease sought to be treated. It is expectedthat the dosages will vary, depending upon the pharmacokinetic andpharmacodynamic characteristics of the particular agent, and its modeand route of administration, as well as the age, weight, and health ofthe recipient; the nature and extent of any relevant disease; thefrequency and duration of the treatment; the type of, if any, concurrenttherapy; and the desired effect.

Any method that accomplishes in vivo transfer of nucleic acids intoeukaryotic cells can be used. For example, expression constructs thereofcan be packaged into liposomes, non-viral nucleic acid-based vectors,erythrocyte ghosts, or microspheres (e.g., microparticles; see, e.g.U.S. Pat. Nos. 4,789,734; and 4,925,673; 3,625,214; and Gregoriadis,Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press,1979)). Further, delivery of nucleic acid therapeutic agents can beaccomplished by direct injection into target tissues, for example, in acalcium phosphate precipitate or coupled with lipids. In certain cases,the nucleic acid therapeutic agents can be modified to increase theirability to penetrate the target tissue by, e.g., coupling them tolipophilic compounds. In addition, nucleic acid therapeutic agents canbe targeted to particular cells by coupling them to ligands specific forreceptors on the cell surface of a target cell. nucleic acid therapeuticagents can also be targeted to specific cell types by being conjugatedto monoclonal antibodies that specifically bind to cell-type-specificreceptors.

For topical administration, a therapeutically effective amount of one ormore of the therapeutic agents is applied to the desired site on theskin, preferably in combination with a pharmaceutically acceptablecarrier, e.g., a spreadable cream, gel, lotion, or ointment, or a liquidsuch as saline. For use on the skin, the penetration of the nucleicacids into the tissue may be accomplished by a variety of methods knownto those of ordinary skill in this field. For example, the expressionconstructs may be incorporated into a transdermal patch that is appliedto the skin. Preferably, the penetration resulting from these methods isenhanced with a chemical transdermal delivery agent such as dimethylsulfoxide (DMSO) or the nonionic surfactant, n-decylmethyl sulfoxide(NDMS), as described in Choi et al., Pharmaceutical Res., 7(11 ):1099,1990. Dosages for a therapeutically effective amount for topicalapplication would be in the range of 100 ng to 10 mg per treated surfacearea per day.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Materials And Methods 1) Vectors, Viral Production, and In VivoTransduction

Replication incompetent lentiviruses used in the gene transferexperiments were created using a five-plasmid transfection procedure.Briefly, 293T cells were transfected using TranIT 293 (Mirus, Madison,Wis.) according to the manufacturer's instructions with a backbonelentiviral vector together with four expression vectors encoding thepackaging proteins gagpol, rev, tat and the G-protein of the vesicularstomatitis virus (VSV). The lentiviral backbone used in the experimentswas a SIN non-replicative vector derived from the original pHR′CMV-lacZvector previously described by Naldini et al (8). The gagpol helperplasmid has been codon-optimized for efficient mammalian expression andmodified to severely reduce the homology with the gag sequences presentin the vector packaging signal. All of the expression helper plasmidscontain only the coding sequences, with minimal 5′ or 3′ untranslatedsequences and no introns. In addition, the backbone contains theWoodchuck Hepatitis virus post-transcriptional regulatory element (WPRE)(9) to enhance levels of transcription and gene expression. Viralsupernatants were collected starting 24 hr after transfection, for fourconsecutive collections every twelve hours, pooled and filtered througha 0.45 μm filter. Viral supenantants were then concentrated ˜100 fold byultracentrifugation in a Beckman centrifuge for 1.5 hr at 16,500 rpm.Using this protocol, titers of ˜5×10⁸-1×10⁹/ml were achieved.

For intratracheal infections, nude mice were anaesthesized and then 100μl of concentrated virus was delivered upon normal inhalation via ablunt-ended 16-gauge needle inserted in such a way to depress thetongue.

2) HSC Purification and Viral Transduction

Purified HSC were obtained by isolating bone marrow SP cells usingfluorescence activated cell sorting after Hoechst staining as previouslydescribed (10), with some modifications. Briefly, femurs and tibias frommice were homogenized, and the bone marrow cells were filtered through70 μm nylon mesh and washed in PBS containing 2% FCS and 0.5% SodiumAzide. Cells were then resuspended in HBSS containing 2% FCS, 10 mMHepes buffer and antibiotics (all from Gibco, Grand Island, N.Y.) and8.8 μg/ml Hoechst 33342 (Molecular Probes, Eugene, Oreg.) at a cellconcentration of 5×10⁶/ml. Following incubation for 90 min at 37° C.,cells were washed once and further purified by using a gradient ofFicoll-Paque™ Plus (Amersham Biosciences AB, Uppsala) to remove redblood cells. Purified marrow cells were then sorted using a MoFlo highspeed cell sorter (DakoCytomation, Fort Collins, Col.). Except whenspecified, cells were kept on ice during the entire procedure. Viraltransduction of sorted HSC was performed using StemPro SFM-34 medium(Gibco) containing 10 ng/ml SCF, 100 ng/ml TPO, and 5 μg/ml polybrene.All cytokines were purchased from R&D Systems (Minneapolis, Minn.).Transduction was performed in a 20 μl reaction for 2.4 hr at 37° C. in awell of a 96-round bottom well plate. Cells were then resuspended in 100μl for transplantation.

3) Bone Marrow Transplantation

All mice were purchased from Jackson Laboratories and maintained in aspecific pathogen-free animal facility at Harvard Medical School. Ly5.2recipient mice were lethally irradiated with 2 doses of 7 Gy, 3 hrapart, one day before BMT and maintained under antibiotic-supplementedwater for 15 days. Transduced SP cells from Ly5.1 donors were injectedretro-orbitally into recipient mice under isofluorane anesthesia. Allanimal procedures were approved by the Standing Committee on Animals ofHarvard Medical School.

4) In vitro Luciferase Assays

FG 293 cells were infected with viral supernatants at a multiplicity ofinfection (MOI) of 10. After 72 hours, cells were then split into sixwell plates, exposed to various concentrations of G1418 for 48 hours,and lysed. Protein extracts were then assayed for luciferase expressionusing the Promega Luciferase Assay System.

5) Non-Invasive Bioluminescent Imaging

Prior to imaging, mice were anaesthesized and injected with 150 μlluciferin (30 mg/ml) (Xenagen, Alameda, Calif.). A series ofbioluminescent images were then taken for up to 30 minutes using theXenogen IVIS imager. Photon output was quantified at the plateau of thetime-course using the Living Image software. Induction in fold wascalculated based on the photon output in the animals before and afterdrug treatment.

Results 1) Design Features of Translation-Based Gene Regulation System

The general strategy for controlling gene expression via the modulationof translational termination is shown in FIG. 1A. To achieve the controlof expression of a specific transgene, a translational termination(‘nonsense’) codon is introduced into transgene coding sequences, closeto the AUG codon that serves to initiate translation of the completeprotein. The modified transgene is then introduced into a standardmammalian expression vector. Upon introduction of the resultingconstruct into cells, translation of the transgene-encoding mRNA resultsin production of only a short, non-functional peptide. Addition of smallmolecules capable of suppressing translational termination to cellsharboring the construct results in production of the full-length desiredprotein.

To prevent the constitutive generation of truncated transgene productsof significant length that might engender immune responses in vivo, wechose to position nonsense codon sequences very close to the initiatorAUG, such that translation termination would result in the production ofa short two to three amino acid peptide, a peptide size insufficient forclassic antigen presentation (11). Such a configuration of sequences wasmodeled after a naturally occurring nonsense mutation in the humanApolipoprotein CII gene (Apo CII_(Paris2)) that results in the nearcomplete loss of the corresponding gene product in a patient carryingthe mutation (12). The relevant nucleotide sequences of wild-type (WT)and mutant forms of the Apo CII gene are shown in FIG. 1B. Since anumber of previous studies had demonstrated that different translationaltermination codons are recognized at different efficiencies, asreflected by the levels of ‘spontaneous’ suppression observed in cells,and that nucleotides directly adjacent to translation terminationsequences can affect termination efficiencies and the extent ofsuppression of termination that can be achieved (13-21), we also choseto evaluate several additional configurations of termination codons andadjacent sequences for both their efficiency of translationaltermination, and their ability to be suppressed by addition of specificcompounds (FIG. 1C and FIG. 3).

Based on previous studies that have shown that aminoglycosideantibiotics are capable of suppressing nonsense mutations in mammaliancell lines and in animal models (13, 14, 19, 20, 22-25), and may actsimilarly in humans (26), a variety of aminoglycoside antibiotics werechosen as potential ‘inducers’ of gene expression and evaluated fortheir relative ability to induce transgene expression via suppression oftranslation termination. G418 (or “Geneticin”) (27) was chosen for ourinitial studies, based on a number of published reports that it was mosteffective aminoglycoside for suppressing nonsense mutation in mammaliancells (13, 19, 23, 25).

2) Aminoglycoside-Induced Suppression of a Stop Codon can be Used toRegulate Gene Expression In Vitro

To facilitate quantitative measurement of both spontaneous read-throughof specific termination codon configurations and the ability of specificantibiotics to suppress translational termination, a luciferase (luc)reporter gene was generated in which the first five codons of either WTmutant MIA-containing) Apo CII sequences were fused to luc codingsequences, and the resulting sequences were introduced into a standardlentiviral vector (FIG. 1B). High-titer virus generated from the vectorswas used to infect human FG293 cells at a multiplicity of infection(MOI) of 10, so that virtually 100% of the cells would be stablytransduced. Cells were then split into six well plates, exposed tovarying concentrations of the aminoglycoside (G418) for 48 hours, andthen lysed and assayed for luc expression. As shown in FIG. 2A, cellstransduced by vectors encoding wild type Apo CII sequences expressedhigh levels of luciferase that were not dependent upon the presence ofantibiotic. In contrast, cells transduced by vectors encoding the mutantApo CII-luc expressed only low levels of luc in the absence of G418(approximately 1.2% the activity of the wild type), but could be inducedto strongly express luc by the addition of the antibiotic (FIG. 2B).Maximum induction of expression was observed in the presence of 300μg/ml of G418, representing an approximately 60-fold increase inreporter expression relative to the induced state. The efficiency ofsuppression of translational termination was extremely high (over 70% ofthe level of wild-type reporter expression observed in the absence ofthe nonsense codon). Northern analysis of RNA isolated from cellstransduced with viruses encoding either WT or mutant Apo CII-luciferasefusion proteins showed that comparable amounts of mRNA were produced,whether or not G418 was present (data not shown). This latter dataindicates that the configuration of initiator and termination codon usedfor gene regulation does not appreciably activate the nonsense-mediatedmRNA decay (NMD) pathway (28). G418 (or “Geneticin”) (27) was chosen forour initial studies, based on a number of published reports that it wasmost effective aminoglycoside for suppressing nonsense mutation inmammalian cells (13, 19, 23, 25).

2) Aminoglycoside-Induced Suppression of a Stop Codon Can Be Used toRegulate Gene Expression In Vitro

To facilitate quantitative measurement of both spontaneous read-throughof specific termination codon configurations and the ability of specificantibiotics to suppress translational termination, a luciferase (luc)reporter gene was generated in which the first five codons of either WTmutant (TGA-containing) Apo CII sequences were fused to luc codingsequences, and the resulting sequences were introduced into a standardlentiviral vector (FIG. 1B). High-titer virus generated from the vectorswas used to infect human FG293 cells at a multiplicity of infection(MOI) of 10, so that virtually 100% of the cells would be stablytransduced. Cells were then split into six well plates, exposed tovarying concentrations of the aminoglycoside (G418) for 48 hours, andthen lysed and assayed for luc expression. As shown in FIG. 2A, cellstransduced by vectors encoding wild type Apo CII sequences expressedhigh levels of luciferase that were not dependent upon the presence ofantibiotic. In contrast, cells transduced by vectors encoding the mutantApo CII-luc expressed only low levels of luc in the absence of G418(approximately 1.2% the activity of the wild type), but could be inducedto strongly express luc by the addition of the antibiotic (FIG. 2B).Maximum induction of expression was observed in the presence of 300μg/ml of G418, representing an approximately 60-fold increase inreporter expression relative to the uninduced state. The efficiency ofsuppression of translational termination was extremely high (over 70% ofthe level of wild-type reporter expression observed in the absence ofthe nonsense codon). Northern analysis of RNA isolated from cellstransduced with viruses encoding either WT or mutant Apo CII-luciferasefusion proteins showed that comparable amounts of mRNA were produced,whether or not G418 was present (data not shown). This latter dataindicates that the configuration of initiator and termination codon usedfor gene regulation does not appreciably activate the nonsense-mediatedmRNA decay (NMD) pathway (28).

3) Kinetics of Induction of Gene Expression UsingAminoglycoside-Mediated Suppression of Translational Termination

One of the expected characteristics of the gene regulation systemdescribed here is the ability to rapidly achieve induction of transgeneexpression, since (as indicated above) transcription is constitutive andtherefore induction of gene expression should depend only upon therestoration of fall translation of the transgene coding sequences. Toaddress this issue, we employed the transduced cells described above,and measured the level of reporter activity after G418 administration(200 μg/ml) as a function of time. As shown in FIG. 2C, as soon as 1hour post induction, the first time period tested, levels of luciferasesignificantly above the levels observed before addition of drug weredetected (1.4% at t=1 hour vs 0.8% wild-type activity at t=0). At 6, 12,and 24 hours post induction, 6.1%, 25.3% and 38.1%, respectively, ofwild-type activity was achieved, with maximal induction noted at 48hours post exposure of the cells to G418 (60-fold induction; 51.3% ofwild-type activity).

4) Basal and Inducible Levels of Transgene Expression are Dependent Uponthe Choice of Termination Codon and Adjacent Sequences

While our initial experiment made use of the precise amino acidsequences represented by mutant human Apo CII NH₂-terminal sequences, wenext asked whether either the basal or induced levels of transgeneexpression could be manipulated through the use of other terminationcodons and surrounding sequences. As shown in FIG. 1C, of all theconfigurations tested, sequences derived from the Apo CII_(Paris2)(TGAC) gene exhibited the greatest spontaneous read-through (1.2%), andthe largest extent of ‘induction’ with aminoglycoside treatment (61-foldinduction, with 73% of WT expression achieved). Interestingly, however,while as expected 19), the ATAAA configuration displayed a significantlylower level of spontaneous read through (0.24%), the levels of inductionthat could be achieved (42-fold) were quite comparable to those achievedwith the TGAC configuration. While the placement of two UGA codonsdirectly adjacent to each other decreased the basal levels of expression(0.39-0.43% of WT expression), the level of induction achieved was onlymoderate (26 to 28 fold). Introduction of sequences between the two UGAcodons to provide a +4 nucleotide previously shown to be permissive toread-through (17) actually led to a further decrease in basal levels(0.11%), and an 18-fold induction. Lastly, the juxtaposition of two UAAcodons led to a low basal level (0.22%) but a poor level of induction(5-fold). These studies indicate that specific configurations oftermination codons and adjacent nucleotide sequences can indeed be usedto provide for different levels of basal and induced gene expression. Itshould be noted from the sequences shown in FIG. 1C that fortuitously,the mutant Apo CII-derived sequences actually encode an additional AUGdirectly overlapping the UGA codon. If translated, a peptide of fouramino acids might be produced in addition to the expected three aminoacid peptide. No fortuitous short open-reading frame is generated as aconsequence of utilizing the TAA-containing configurations.

5) G418 is the Most Effective Aminoglycoside Inducer of Gene Expression

While several previous studies had strongly suggested that G418 was moreeffective at suppressing nonsense mutations than other aminoglycosides23, 25), we sought to confirm this result in the context of theluciferase-based gene regulation assay described above. Compounds weretested in vitro using stable cell lines generated via the infection ofFG293 cells with lentiviral vectors carrying either WT or mutant Apo CIIluc sequences. The aminoglycosides were tested over a concentrationrange from 0 to 1000 μg/ml. As shown in FIG. 3A, none of the otheraminoglycosides tested (gentamicin, amakacin, tobramycin, andparomomycin) were comparable to G418 in their ability to induceluciferase expression, as they displayed only weak induction throughoutthe aminoglycoside concentration range tested.

Further, two novel compounds were found to be effective in suppressingtranslation termination codons FG293 cells. FIG. 3B indicates that thefold of induction mediated by3-[2-(4-tert-butyl-phenoxyl)-acetylamino]-benzoic acid was about 14% atthe concentration of 5 μg/ml. FIG. 3C indicates the fold of inductionmediated by 3-{2-[4-(1,1-dimethyl-propyl)-phenoxyl]acetylamino}-benzoicacid was about 24% at the concentration of 5 μg/ml. Both compounds werepurchased from Chembridge.

6) Aminoglycoside Induced Suppression of Translational Termination canRegulate Gene Expression In Vivo

To further establish the general applicability of the gene retaliationsystem, we next asked whether gene regulation at the level oftranslation could be readily accomplished in the in vivo setting. In afirst animal model, intratracheal delivery (see Materials and Methods)of a lentiviral vector carrying WT or mutant Apo CII-luc sequences wasutilized in order to assess the ability to regulate luc expression inmurine lung tissue via aminoglycoside administration. Five days posttransduction, prior to administration of aminoglycoside, mice wereimaged for luc expression using the Xenogen IVIS non-invasivebioluminescent imager (29). In contrast to the animals infected with thevector encoding WT Apo CII luc Sequences (WT luc mice), which displayeda strong photon signal indicative of luciferase activity, animals thatwere infected with the vector encoding mutant Apo CII-luc sequences(mutant luc mice) showed no detectable signal (data not shown). Micewere then injected for 4 days, once daily, with either G418 or PBS,except that on the fourth day when mice were injected twice, once in themorning and then again 60 minutes before luciferase imaging. The dailydose of G418 chosen for administration was 1.5 mg, an amount whichrepresents approximately half of the published LD₅₀ for G418 (25). Asexpected, upon imaging, WT luc mice showed strong luc expression,whether or not G418 had been administered. In contrast, while mutant lucmice that were injected with PBS continued to display no detectableluciferase activity, mutant luc mice that were subsequently administeredG418 showed significant induction of luciferase activity. The five micetreated in this way displayed significant luciferase expression,resulting in an average of 27% of the level of expression observed in WTluc mice. Due to the lack of detectable luc expression in uninducedmice, it was not possible to determine a ‘fold-induction’.

To further evaluate the potential in vivo utility of the gene regulationsystem, a standard murine bone marrow transplantation model was used toassess the ability to regulate genes introduced into hematopoietic cellsvia lentiviral vectors. For these studies, 500 purified C57/B16hematopoietic stem cells (SP cells) (10) were transduced with lentiviralvectors encoding either wild-type or mutant reporters and thenintroduced into lethally-irradiated recipients. Four weeks after bonemarrow transplantation, reconstituted animals were imaged for luciferaseexpression and then subsequently treated with PBS or G418 daily for 3.5days (1.5 mg/day). Prior to PBS or G418 administration, animalsreconstituted with cells transduced by virus encoding mutant Apo CII lucsequences (mutant luc mice) showed negligible luc expression, whileanimals reconstituted with cells transduced by viruses encoding WT ApoCII luc sequences (WT luc mice) showed robust luc expression (data notshown). After PBS or G418 administration, WT luc mice continued to showstrong luc expression. While PBS administration had no effect on lucexpression in mutant luc mice, antibiotic treatment of those mice led tothe strong induction of luc. Quantitation of luciferase activity in fourmutant luc animals before and after drug administration demonstrated anaverage 65-fold induction of luc expression, with induced levelsapproaching an average of 51% of the expression observed in WT luc mice.

7) Aminoglycoside Induced Suppression of Translational Termination CanEffectively Regulate Gene Expression and Secretion of Human GrowthHormone (hGH).

FIG. 4 presents data that shows that G418 effectively induces geneexpression of human growth hormone gene (hGH) by suppressing translationtermination codons, resulting in increased secretion of hGH protein asmeasured by ELISA.

Discussion

The studies reported here demonstrate the feasibility of utilizingtranslation-based systems for the control of gene expression inmammalian cells both in vitro and in vivo. Perhaps the most importantdesign feature of this gene regulation strategy is its remarkablesimplicity: the only ‘genetic element’ required is an approximatelythree to ten nucleotide DNA sequence encoding a translationaltermination codon and in some cases, additional sequences. Because theregulatory element resides within transgene coding sequences, it shouldbe possible to achieve regulation in the context of virtually anyexpression vector, and to provide for the regulation of expression ofprotein coding sequences with the context of their normal endogenouscontrol elements. In addition, since we have shown that, at least in thecase of the configuration of initiation and termination codons tested,the production of mRNA is constitutive; induction of gene expressionshould be dependent only on the restoration of translation of thecomplete coding sequences. Therefore, the kinetics of induction of geneexpression would be expected to be more rapid than gene regulationsystems based on the control of transcription or mRNA cleavage, in whichcases RNA half-life is a relevant variable.

The conception and design of the translation-based gene regulationsystem described above was strongly guided by a large number of previousstudies which have focused on the phenomenon of translational‘miscoding’ in mammalian cells (30). Those studies have provided adetailed understanding of both the local sequence contexts that affectthe extent of spontaneous read-through of different termination codons,and the ability or specific aminoglycoside antibiotics to enhance suchread-through (13-21). The known sequence determinants governing theefficiency of the spontaneous read-through of termination codons wereparticularly germaine to our studies, in so far as they enabled thedesign of regulatory elements that provided for different levels of‘basal’ and ‘induced’ gene expression, an important practical capabilityof the strategy. While our studies explored only a small number ofpermutations of sequences encoding different termination codons andadjacent sequences, the available literature suggests that parameters ofregulation such as basal and induced levels of expression could be veryprecisely manipulated by the empirical evaluation of a wide range ofdifferent sequence configurations.

In the case of the present studies, the positioning of the nonsensecodon relative to transgene coding sequences was based upon theexistence of a naturally occurring mutation in the human Apo CII gene(Apo CII_(Paris2)) known to dramatically affect production of the normalgene product in a patient carrying the mutation (12). The closejuxtaposition of initiation and termination codons, represented by theApo CII mutation, was thought to an important design feature of thetranslation-based regulation system, since the small peptide resultingfrom the premature termination of translation initiating from theauthentic AUG would be too small to enter the classic pathway forantigen presentation (11). Clearly, however, as was pointed out earlierwith regard to the Apo CII sequences used in our studies, depending uponthe specific coding sequences of the transgene, there may be thepossibility of producing other truncated peptides. In such cases,additional modifications of sequences may be warranted.

Another benefit of the placement of the termination codon close to thebeginning of protein-coding sequences shown by our studies is that thisspecific configuration did not lead to any appreciatiable reduction ofmRNA levels due to activation of the NMD pathway (28), a response thatmight compromise either the extent or kinetics of ‘induction’ of geneexpression. Our data is consistent with a very recent study which showedthat a naturally occurring mRNA which possessed a nonsense mutationlocated close to the initiator AUG was not subject to nonsense-mediatedmRNA decay (NMD) (31).

Lastly, with regard to the placement of nonsense codons adjacent to theinitiator AUG, it is noteworthy that in the case of severalconfigurations of sequences we evaluated, it was possible to obtainextremely high levels of suppression of translation by aminoglycosideadministration, in some cases over 70% the levels of wild-type geneexpression. As such high levels of suppression have not been previouslyreported by others, it is possible that the placement of terminationcodons close to the initiator codon may uniquely facilitate thesuppression of translational termination by aminoglycosides. Futureexperiments will address this issue as well as the role, if any, of thestrength of the termination codon at the end of transgene protein codingsequences in determining suppression efficiencies.

One limitation of the design of the system is that introduction of the‘regulatory element’ into transgene sequences can alter the amino acidsequence of the resulting gene product. While for many experimentalapplications, slight alterations in amino acid sequences may beirrelevant, in certain cases, regulation of an authentic gene productmay be critical. This limitation may be addressed in several ways.First, depending on the native amino acid sequence of the protein ofinterest, it may be possible to design configurations in whichsubstitution of a single amino acid codon with a specific terminationcodon is sufficient for gene regulation, taking account of knowledgeregarding the specific amino acids that are inserted when specificnonsense codons are misread (32) and the effects of nucleotides directlyadjacent to termination codons on the efficiency of termination (17,19). In this regard, we have shown that it was possible to achieveefficient gene regulation of a gene product through substitution of onlytwo amino acid codons present in a coding sequence (unpublishedresults). In that particular case, the termination codon fortuitouslyreplaced an amino acid codon specifying the same amino acid as thatwhich would be predicted to be introduced by misreading of thetermination codon (32). Second, in the special case of regulating theexpression of secreted gene products, in which the mature gene productis generated by co-translational cleavage of the pre-protein (33),modifications of sequences adjacent to the initiator AUG should notaffect the structure of the mature gene product and therefore it shouldgenerally be possible to regulate expression of an authentic secretedproduct. In recent studies, we have documented the ability to providefor the regulated expression of a secreted gene product, through theintroduction of a nonsense codon into ‘signal peptide’ encodingsequences (unpublished results).

Overall, the system described here represents a remarkably simple meansof controlling the expression of genes in mammalian cells that shouldhave immediate experimental applications. While the range ofinducibility obtainable with the system (in some cases over 60 fold) isconsiderably more limited than that of gone regulation systems based onthe control of transcription or RNA self-cleavage, the ability tofine-tune the basal and induced levels of gene expression via use ofspecific termination codons and adjacent sequences may mitigate thislimitation in many experimental situations. With regard to the issue ofthe availability of suitable inducers for common experimentalapplications, it is ironic that we and other have shown that the mosteffective aminoglycoside for suppressing nonsense mutations in mammaliancells is G418 (Geneticin), an antibiotic most well known to molecularbiologists for its ability to cells in the context of selections fortransduced cells (34). Despite this ‘reputation’, we have shown herethat G418 can be effectively utilized to regulate gene expression in anumber of cell lines and in animals at concentrations that are notacutely toxic. Similarly, others have shown that G418 can be used atrelatively non-toxic doses to suppress nonsense mutations in cell linesand in animal models of inherited disease (25, 35). These results maylend support to the notion that the requirements for the suppression ofnormal translational termination at the end of protein coding sequencesmay be more stringent than those important for the suppression ofnonsense mutations. Furthermore, based on a number of animal studiesthat suggest the possibility that the suppression of nonsense mutationscould provide a novel therapeutic approach to the treatment of certaininherited diseases (19, 22-25, 36), there have been intense efforts toidentify non-toxic small molecules suitable for use in humans. Theavailability of such molecules for experimental studies in the futureshould further expand the utility of translation-based gene regulationstrategies.

References

-   1. Gossen, M, & Bujard, H. (1992) Proc Natl Acad Sci USA 89,    5547-51.-   2. Wang, Y., O'Malley, B. W., Jr., Tsai, S. Y. & O'Malley B.    W, (1994) Proc Natl Acad Sci USA 91, 8180-4.-   3. Rivera, V. M., Clackson, T., Natesan, S., Pollock, R., Amara, J.    F., Keenan, T., Magari, S. R., Phillips, T., Courage, N. L.,    Cerasoli, F., Jr., Holt, D. A. & Gilman, M. (1996) Nat Med 2,    1028-32.-   4. Suhr, S. T., Gil, E. B., Senut, M. C. & Gage, F. H. (1998) Proc    Natl Acad Sci USA 95, 7999-8004.-   5. Gossen, M., Bonin, A. L., Freundlieb, S. & Bujard, H. (1994) Curr    Opin Biotechnol 5, 516-20.-   6. Rivera, V. M., Gao, G. P., Grant, R. L., Schnell, M. A.,    Zoltick, P. W., Rozamus, L. W., Clackson, T. & Wilson J. M. (2005)    Blood 105, 1424-30.-   7. Yen, L., Svendsen, J., Lee, J. S., Gray, J. T., Magnier, M.,    Baba, T., D'Amato, R. J. & Mulligan, R. C. (2004) Nature 431, 471-6.-   8. Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R.,    Gage, F. H., Verma, I. M. & Trono, D., (1996) Science 272, 263-7.-   9. Zufferey, R., Donello, J. E., Trono, D. & Hope, T. J. (1999) J.    Virol 73, 2886-92.-   10. Goodell, M. A., Brose, K., Paradis, G., Conner, A. S. &    Mulligan, R. C. (1996) J Exp Med 183, 1797-806.-   11. Rammensee, H. G., Falk, K. & Rotzschke, O., (1993) Annu Rev    Immunol 11, 213-44.-   12. Parrott, C. L., Alsayed, N., Rebourcet, R. &    Santamarina-Fojo, S. (1992) J Lipid Res 33, 361-7.-   13. Burke, J. F. & Mogg, A. E. (1985) Nucleic Acids Res 13, 6265-72.-   14. Martin, R., Mogg, A. E., Heywood, L. A., Nitschke, L. &    Burke, J. F. (1989) Mol Gen Genet 217, 411-8.-   15. Brown, C. M., Stockwell, P. A., Trotman, C. N. &    Tate, W. P. (1990) Nucleic Acids Res 18, 6339-45.-   16. Martin, R. (1994) Nucleic Acids Res 22, 15-9.-   17. McCaughan, K. K., Brown, C. M., Dalphin, M. E., Berry, M. J. &    Tate, W. P. (1995) Proc Natl Acad. Sci USA 92, 5431-5.-   18. Phillips-Jones, M. K., Hill, L. S., Atkinson, J. &    Martin, R. (1995) Mol Cell Biol 15, 6593-600.-   19. Howard, M. T., Shirts, B. H., Petros, L. M., Flanigan, K. M.,    Gesteland, R. F. & Atkins, J. F. (2000) Ann Neurol 48, 164-9.-   20. Manuvakhova, M., Keeling, K. & Bedwell, D. M. (2000) Rna 6,    1044-55.-   21. Namy, O., Hatin, I. & Rousset, J. P. (2001) EMBO Rep 2, 787-93.-   22. Barton-Davis, E. R., Cordier, L., Shoturma, D. L., Leland, S. E.    & Sweeney, H. L. (1999) J. Clin Invest 184, 375-81.-   23. Howard, M. T., Anderson, C. B., Fass, U., Khatari, S.,    Gesteland, R. F., Atkins, J. F. & Flanigan, K. M. (2004) Ann Neurol    55, 422-6.-   24. Hein, L. K., Bawden, M., Muller, V. J., Sillence, D.,    Hopwood, J. J. & Brooks, D. A. (2004) J Mol Biol 338, 453-62.-   25. Sankuhl, K. Schulz, A. Rompler, H., Yun, J., Wess, J. &    Schoneberg, T. (2004) Hum Mol Genet 13, 893-903.-   26. Wilschanski, M., Yahav, Y., Yaacov, Y., Blau, H., Bentur, L.,    Rivlin, J., Aviram, M., Bdolah-Abram, T., Bebok, Z., Shushi, L.,    Kerem, B. & Kerem, E. (2003) N Engl J. Med 349, 1433-41.-   27. Loebenberg, D., Counels, M. & Waitz, J. A. (1975) Antimicrob    Agents Chemother 7, 811-5.-   28. Frischmeyer, P. A. & Dietz, H. C. (1999) Hum Mol Genet 8,    1893-900.-   29. Contag, C. H. & Bachmann, M. H. (2002) Anna Rev Biomed Eng 4,    235-60.-   30. Gesteland, R. F. Weiss, R. B. & Atkins, J. F. (1992) Science    257, 1640-1.-   31. Inacio, A., Silva, A. L., Pinto, J., Ji, X., Morgado, A.,    Almeida, F., Faustino, P., Lavinha, J., Liebhaber, S. A. &    Romao, L. (2004) J Biol Chem 279, 32170-80.-   32. Nilsson, M. & Ryden-Aulin, M. (2003) Biochim Biophys Acta 1627,    1-6.-   33. Rothman, J. E. (1994) Nature 372, 55-63.-   34. Colbere-Garapin, F., Horodniceanu, F., Kourilsky, P. &    Garapin, A. C. (1981) J Mol Biol 150, 1-14.-   35. Lai, C. H., Chun, H. H., Nahas, S. A., Mitui, M., Gamo, K. M.,    Du, L. & Gatti R. A. (2004) Proc Natl Acad Sci USA 101, 15676-81.-   36. Howard, M., Frizzell, R. A. & Bedwell, D. M. (1996) Nat Med 2,    467-9.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The scope ofthe invention should be determined by reference to the claims, alongwith their full scope of equivalents, and the specification, along withsuch variations.

1.-62. (canceled)
 63. A recombinant polynucleotide molecule comprising anucleic acid encoding a target protein, wherein the nucleic acid isoperably linked to a promoter and has been modified to contain anonsense codon, wherein the number of codons between the start codonthat initiates translation of the target protein and the nonsense codonis 0, 1, 2, 3, 4, or
 5. 64. The polynucleotide molecule of claim 63,wherein the nonsense codon is TGA or TAA.
 65. The polynucleotidemolecule of claim 64, wherein the nonsense codon is TGA.
 66. Thepolynucleotide molecule of claim 64, wherein the nonsense codon is TAA.67. The polynucleotide molecule of claim 63, wherein the nucleotidefollowing the nonsense codon is C or A.
 68. The polynucleotide moleculeof claim 67, wherein the nucleotide following the nonsense codon is C.69. The polynucleotide molecule of claim 67, wherein the nucleotidefollowing the nonsense codon is A.
 70. The polynucleotide molecule ofclaim 63, wherein the target protein comprises a signal peptide.
 71. Thepolynucleotide molecule of claim 70, wherein the target protein is asecreted protein.
 72. The polynucleotide molecule of claim 71, whereinthe nonsense codon is introduced in a region of the nucleic acid whichencodes the signal peptide.
 73. The polynucleotide molecule of claim 63,wherein the polynucleotide molecule is present in the genome of a cell.74. The polynucleotide molecule of claim 63, wherein the polynucleotidemolecule is present on a vector.
 75. The polynucleotide molecule ofclaim 74, wherein the vector is a viral vector.
 76. A host cellcomprising the polynucleotide molecule of claim
 63. 77. The host cell ofclaim 76, wherein the cell is a mammalian cell.
 78. The host cell ofclaim 76, wherein the cell is a mammalian cell and further comprises anagent which suppresses the nonsense mutation.
 79. A recombinant vectorfor inducible expression of a target protein comprising: a) a promoter;b) a nucleic acid comprising a start codon and encoding a linkerpeptide, wherein the nucleic acid is operably linked to the promoter andhas been modified to contain a nonsense codon; and c) at least onecloning site downstream to the nucleic acid for introducing a targetnucleic acid sequence which encodes a target protein to be fused inframe to the carboxyl terminus of the linker peptide; wherein the numberof codons between the start codon and the nonsense codon is 0, 1, 2, 3,4, or
 5. 80. The vector of claim 79, wherein the nonsense codon is TGAor TAA.
 81. The vector of claim 80, wherein the nonsense codon is TGA.82. The vector of claim 80, wherein the nonsense codon is TAA.
 83. Thevector of claim 79, wherein the nucleotide following the nonsense codonis C or A.
 84. The vector of claim 83, wherein the nucleotide followingthe nonsense codon is C.
 85. The vector of claim 83, wherein thenucleotide following the nonsense codon is A.
 86. The vector of claim79, wherein the linker peptide is a signal peptide.
 87. The vector ofclaim 86, wherein the signal peptide is a native or a non-native signalpeptide of the target protein.